Inhibition of Perforin-Mediated Neurotoxicity Attenuates Neurological Deficits After Ischemic Stroke

Abstract

Perforin-mediated cytotoxicity plays a crucial role in microbial defense, tumor surveillance, and primary autoimmune disorders. However, the contribution of the cytolytic protein perforin to ischemia-induced secondary tissue damage in the brain has not been fully investigated. Here, we examined the kinetics and subpopulations of perforin-positive cells and then evaluated the direct effects of perforin-mediated cytotoxicity on outcomes after ischemic stroke. Using flow cytometry, we showed that perforin⁺CD45⁺ immune cells could be detected at 12 h and that the percentage of these cells increased largely until on day 3 and then significantly declined on day 7. Surprisingly, the percentage of Perforin⁺CD45⁺ cells also unexpectedly increased from day 7 to day 14 after ischemic stroke in Perforin1-EGFP transgenic mice. Our results suggested that Perforin⁺CD45⁺ cells play vital roles in the ischemic brain at early and late stages and further suggested that Perforin⁺CD45⁺ cells are a heterogeneous population. Surprisingly, in addition to CD8⁺ T cells, NK cells, and NKT cells, central nervous system (CNS)-resident immune microglia, which are first triggered and activated within minutes after ischemic stroke in mice, also secreted perforin during ischemic brain injury. In our study, the percentage of perforin⁺ microglia increased from 12 h after ischemic stroke, increased largely until on day 3 after ischemic stroke, and then moderately declined from days 3 to 7. Intriguingly, the percentage of perforin⁺ microglia also dramatically increased from days 7 to 14 after ischemic stroke. Furthermore, compared with wild-type littermates, Perforin 1^-/- mice exhibited significant increases in the cerebral infarct volume, neurological deficits, and neurogenesis and inhibition of neurotoxic astrogliosis. Interestingly, the number of CD45⁺CD3⁺ T cells was significantly decreased in Perforin 1^-/- mice compared with their wild-type littermates, especially the number of γδ T cells. In addition, Perforin 1^-/- mice had lower levels of IL-17 than their wild-type littermates. Our results identified a critical function of perforin-mediated neurotoxicity in the ischemic brain, suggesting that targeting perforin-mediated neurotoxicity in brain-resident microglia and invading perforin⁺CD45⁺ immune cells may be a potential strategy for the treatment of ischemic stroke.

Keywords: cytotoxicity; gliogenesis; immunity; ischemic stroke; microglia; neurogenesis; perforin.